Abstract A person, usually a child, dies of rabies every nine minutes globally. Current rabies virus (RABV) post- exposure prophylaxis (PEP) remains complicated and costly, requiring four to five doses of inactivated RABV-based plus rabies immune globulin (RIG) to stunt the virus while vaccine-induced antibodies are forming. A single-dose vaccine that does not require expensive RIG would enhance the efficacy of RABV vaccination, reduce the cost associated with rabies prevention, and save lives. For the last 35 years, B cells secreting IgG (but not IgM) were thought to be solely responsible for vaccine-induced protection against RABV infection via T cell-dependent (TD) responses in PEP settings. However, we showed that vaccine- induced T cell-independent (TI) and early extrafollicular TD B cell responses, including neutralizing IgM, can limit dissemination of pathogenic RABV into the CNS, partially protecting mice. We also showed that ?free? RABV particles migrate to subcapsular sinus macrophages in the draining lymph node, transferring RABV antigen directly to B cells. Finally, we showed that displaying membrane-anchored molecular adjuvants on the surface of the RABV particle enhances the kinetics and magnitude of the early B cell response compared with RABV particles not displaying the molecular adjuvant. We aim to exploit our findings by developing novel RABV-based virus like particles (VLPs). Specifically, we hypothesize that the highly ordered and repetitive display of proteins on the surface of RABV-based VLPs will provide an excellent scaffold to display antigen (RABV glycoprotein) and a membrane-anchored molecular adjuvant directly to B cells. The extensive cross-linking of RABV-based VLPs to activating receptors on antigen-specific B cells will result in B cells rapidly differentiating into IgM and IgG plasma cells, enhancing the kinetics and magnitude of RABV-specific humoral immunity. In this proposal, we will construct two RABV-based VLPs and then determine the relative potency of each. The VLP with the highest relative potency will be selected as the backbone to insert virus membrane-anchored molecular adjuvants. Next, the new VLPs will be tested in well-described mouse models of rabies immunogenicity and post-exposure protection. The milestone for this project is the identification of a safe RABV-based VLP that induces potent and rapid TI and extrafollicular TD B cell responses more effectively than the multi-dose human rabies vaccine, without the need for RIG, for future investigation. This project also addresses mechanisms important for other antibody-based vaccines under development: i.e., how can viral vaccines target B cell to induce rapid, potent and long-lasting B cell responses using only a single, low-dose of a VLP-based vaccine? In summary, single-dose vaccine strategies capable of eliciting rapid and robust B cell responses will improve the efficacy of human rabies vaccines, reduce the cost associated with rabies prevention and save lives.